Not applicable.
Cellulases are enzymes that are capable of hydrolysis of the xcex2-D-glucosidic linkages in celluloses. Cellulolytic enzymes have been traditionally divided into three major classes: endoglucanases, exoglucanases or cellobiohydrolases and xcex2-glucosidases (Knowles, J. et al., (1987), TIBTECH5, 255-261); and are known to be produced by a large number of bacteria, yeasts and fungi.
Although cellulases are used to degrade wood pulp and animal feed, cellulases are primarily used in the treatment of textiles, e.g., in detergent compositions for assisting in the removal of dirt or grayish cast (see e.g., Great Britain Application Nos. 2,075,028, 2,095,275 and 2,094,826) or in the treatment of textiles prior to sale to improve the feel and appearance of the textile. Thus, Great Britain Application No. 1,358,599 illustrates the use of cellulase in detergents to reduce the harshness of cotton containing fabrics.
Cellulases have also been used in the treatment of textiles to recondition used fabrics by making their colors more vibrant (see e.g., The Shizuoka Prefectural Hammamatsu Textile Industrial Research Institute Report 24:54-61 (1986)). Repeated washing of cotton containing fabrics results in a grayish cast to the fabric which is believed to be due to disrupted and disordered fibrils, sometimes called xe2x80x9cpillsxe2x80x9d, caused by mechanical action. This greyish cast is particularly noticeable on colored fabrics. As a consequence, the ability of cellulase to remove the disordered top layer of the fiber and thus improve the overall appearance of the fabric has been of value.
Because of its effectiveness in many industrial processes, there has been a trend in the field to search for specific cellulase compositions or components that have particularly effective performance profiles with respect to one or more specific applications. As possible sources of cellulases, practitioners have focused on fungi and bacteria. For example, cellulase produced by certain fungi such as Trichoderma spp. (especially Trichoderma reesei) have been given much attention because a complete cellulase system capable of degrading crystalline forms of cellulose is readily produced in large quantities via fermentation procedures. This specific cellulase complex has been extensively analyzed to determine the nature of its specific components and the ability of those components to perform in industrial processes (see, Wood et al., xe2x80x9cMethods in Enzymologyxe2x80x9d, 160, 25, pages 234, et seq. (1988). U.S. Pat. No. 5,475,101 (Ward et al.) discloses the purification and molecular cloning of one particularly useful enzyme called endoglucanase III (EGIII) which is derived from Trichoderma reesei. 
PCT Publication No. WO 94/14953 discloses endoglucanases that are encoded by a nucleic acid that comprises any one of a series of DNA sequences, each having 20 nucleotides.
Ooi, et al., Curr. Genet. 18:217-222 (1990) disclose the cDNA sequence coding for endoglucanase F1-CMC produced by Aspergillus aculeatus that contains the amino acid strings NNLWG, ELMIW and GTEPFT. Sakamoto, et al., Curr. Genet. 27:435-439 (1995) discloses the cDNA sequence encoding the endoglucanase CMCase-1 From Aspergillus kawachii IFO 4308 which contains the amino acid strings ELMTW and GTEPFT. Ward, et al., discloses the sequence of EGIII having the amino acid strings NNLWG, ELMIW and GTEPFT. Additionally, two cellulase sequences, one from Erwinia carotovara and Rhodothermus marinus are disclosed in Saarilahti, et al., Gene 90:9-14 (1990) and Hreggvidsson, et al., Appl. Environ. Microb. 62:3047-3049 (1996) that contain the amino acid string ELMIW.
Despite knowledge in the art related to many cellulase compositions having applications in some or all of the above areas, there is a continued need for new cellulase compositions which have improved stability under conditions present in applications for which cellulases are useful, e.g., household and laundry detergents and textile treatment compositions.
A variant EGIII cellulase is provided wherein the variant comprises a substitution at a residue that is sensitive to surfactant and/or temperature stress and is derived from T. reesei EGIII cellulase. In a preferred embodiment, the variant comprises a substitution or deletion at a position corresponding to one or more of residues W7, T11, T16, A35, S39, G41, S63, A66, S77, N91, S143, T163, N167 and/or, A188. In a more preferred embodiment, the variant comprises a substitution at a position corresponding to one or more of residues W7Y, T11S, T16I, A35S, S39N, G41A, S63V, A66N, S77G, N91D, S143T, T163S, N167S and/or, A188G.
In another embodiment of the invention, a DNA encoding the variant EGIII cellulase is provided. In a preferred aspect of this embodiment, the DNA is in a vector. In another aspect of this embodiment, the vector is used to transform a host cell.
In yet another embodiment, a method of producing a variant EGIII cellulase having improved stability and/or performance is provided. The method comprises the steps of culturing a host cell in a suitable culture medium under suitable conditions to produce cellulase and obtaining the produced cellulase. In another embodiment a detergent composition comprising a surfactant and a variant EGIII cellulase is provided, wherein the variant EGIII cellulase comprises a substitution at residue sensitive to surfactant and/or temperature. In a preferred embodiment, the variant comprises a substitution or deletion at a position corresponding to one or more of residues W7, T11, T16, A35, S39, G41, S63, A66, S77, N91, S143, T163, N167 and/or, A 188G. In a more preferred embodiment, the variant EGIII cellulase comprises a substitution at a position corresponding to one or more of residues W7Y, T11S, T16I, A35S, S39N, G41A, S63V, A66N, S77G, N91D, S143T, T163S, N167S and/or, A188G. In another aspect of this embodiment, the detergent is a laundry or a dish detergent.
In another embodiment of this invention, the variant EGIII cellulase is used in the treatment of a cellulose-containing textile, preferably to stone wash indigo dyed denim. In another embodiment, the variant is used as a feed additive. In yet another embodiment, the variant is used in the treatment of wood pulp. In still another embodiment, the variant is used in the reduction of biomass to glucose.